Worldwide efforts to determine the complete nucleotide sequence of the human genome as well as the genomes of other organisms will result in a huge catalog of sequence information. The goals of the human genome program are to have the project completed by early in the first decade of the 21st century. A major challenge which follows is to identify the functions of the tens of thousands of open reading frames that will result.
One of the technologies which will be utilized for this is antisense-, ribozyme- or DNAzyme-mediated destruction of the sequences, followed by phenotypic and more detailed physiologic and biochemical analyses of cells in which the target has been destroyed. A major problem confronting all antisense technologies is that of rapid throughput screening of sites accessible to pairing by the antisense DNAs and RNAs.
Several approaches have been described to determine the accessibility of a target RNA molecule to antisense or ribozyme inhibitors. One approach uses an in vitro screening assay applying as many antisense oligodeoxynucleotides (antisense ODNs) as possible. Monia et al., Nature Med. 2:668–675 (1996); Milner et al., Nature Biotechnol. 15:537–541 (1997). Another utilizes random libraries of ODNs. Ho et al., Nucleic Acids Res. 24:1901–1907 (1996)(Ho et al. I); Birikh et al., RNA 3:429–437 (1997); Lima et al., J. Biol. Chem. 272:626–638 (1997). The accessible sites can be monitored by RNase H cleavage. Birikh et al, supra; Ho et al., Nature Biotechnol. 16:59–63 (1998) (Ho et al., II). RNase H catalyzes the hydrolytic cleavage of the phosphodiester backbone of the RNA strand of a DNA-RNA duplex.
A pool of semi-random, chimeric chemically synthesized ODNs have been used to identify accessible sites cleaved by RNase H on an in vitro synthesized RNA target. Primer extension analyses were used to identify these sites in the target molecule. Lima et al., supra. Other approaches for designing antisense targets in RNA are based upon computer assisted folding models for RNA. Several reports have been published on the use of random ribozyme libraries to screen effective cleavage. Campbell et al., RNA 1:598–609 (1995); Lieber et al., Mol. Cell Biol. 15, 540–551 (1995); Vaish et al., Biochemistry 36:6459–6501 (1997).
In vitro approaches which utilize random or semi-random libraries of ODNs and RNase H seem to be more useful than computer simulations. Lima et al., supra. However, use of in vitro synthesized RNA does not provide satisfactory results for predicting the accessibility of antisense ODNs in vivo. Recent observations suggest that annealing interactions of polynucleotides are influenced by RNA-binding proteins. Tsuchihashi et al., Science 267:99–102 (1993); Portman et al., EMBO J. 13:213–221 (1994); Bertrand and Rossi, EMBO J. 13:2904–2912 (1994). It is therefore important to utilize cellular RNA-binding proteins in assays for elucidating RNA target accessibility to antisense, ribozyme or DNAzyme binding.